1. Field of the Invention
This invention relates to a novel Endo-deoxyribonuclease (hereinafter referred to as "Endo-DNase") and a process for the production thereof and more particularly, a novel Endo-deoxyribonuclease A (hereinafter referred to as "the Endo-DNase A" or "the DNase A") which has the substrate specificity of recognizing specific base sequence in double-stranded deoxyribonucleic acid (DNA) molecules and cleaving the strands at specified sites in the DNA to produce specific DNA fragments and, a process for the production of the Endo-DNase A which comprises culturing an Endo-DNase A-producing microorganism belonging to the genus Saccharomyces or Pichia and treating the resulting cell-free extract to separate and collect the Endo-DNase A.
The term "novel Endo-deoxyribonuclease A (Endo-DNase A)" means an enzyme according to the present invention and has physico-chemical properties and a substrate specificity which are explained hereinafter.
Deoxyribonuclease (DNase) which degrades deoxyribonucleic acid (DNA) exists in various biological materials, and is involved the important processes of vital functions including DNA metabolism, such as degradation, synthesis and genetic recombination of DNA and public attention has recently been paid to its enzymatic chemical properties and biochemical functions. On the other hand, it has become of great importance that an enzyme having specific functions be produced and separated in order to study the structure and function of gene DNA and to use the enzyme as a biochemical reagent, especially means for gene cloning, for the purpose of breeding.
2. Description of the Prior Art
DNases are generally classified into exonucleases and endonucleases according to their mode of action. The former type acts on the terminal of polynucleotide chain of DNA molecule and hydrolyzes the chain progressively to liberate nucleotides, while the latter cleaves phosphodiester bond in DNA molecule distributively to produce DNA fragments or oligonucleotides. Recently, in the field of endonuclease type enzymes, many studies have been carried out with respect to enzymes having a specificity to the structure of DNA, particularly to the nucleotide sequence or the structural change which exists in nature or is artificially introduced, enzymes recognizing and acting on DNA of a specific organism, or enzymes having biologically important functions (see Tadahiko Ando, Chemistry and Life, Vol. 13, No. 6, p. 342, (1975)).
The inventors of the present invention have performed a series of studies with respect to processes for the production of enzymes and have established some processes which are: a process for producing an enzyme which preferentially cleaves purine-purine linkage in DNA molecule from the cell of Aspergillus oryzae (Japanese Pat. No. 621,205); a process for production an enzyme which preferentially cleaves guanine-guanine linkage in DNA molecule from the culture liquid of alkalophilic bacteria (Japanese Pat. No. 831,171); and, a process for producing three Endo-DNases which recognize the DNA of a specific organism and cleave specific sites in the DNA molecule to form DNA fragments of specific size from the cells of Sporogenous aerobic belonging to the genus Bacillus (Japanese Pat. No. 1,008,416).
Studies of DNase which recognizes the feature of the primary (nucleotide sequence) or high order structure of DNA and acts on the DNA, have recently been directed to clarification of functions of DNA in important processes in cells such as DNA replication, genetic recombination, DNA repair or restriction and modification, and to research and use of enzymes which may be utilized to analysis of DNA structure, formation of site-specific mutagenesis, process for genetic recombination of DNA in vitro, and the like.
Some site-specific endonuclease which recognize specified base sequences in DNA molecules and cleave the strands at specified sites within or near the sequence to produce specific DNA fragments, that is, the restriction enzyme (II) which concerns restriction and modification in procaryotes, and the other various enzymes having similar functions to the enzyme (II) have been found and many of them have been isolated (see Tadahiko Ando, "Restriction enzyme", Chemistry and Life, Vol. 17, No. 5, p. 311, (1979); Tadahiko Ando, "What is a restriction enzyme?--Specificity of restriction enzyme and use thereof", Chemistry, Vol. 35, No. 1, p. 20, (1980)).
However, no enzymes of the type described above have ever been discovered in eucaryotes. The inventors of the present invention conducted the study of a new DNase having the specific function described above with respect to various yeasts belonging to eucaryotes and have succeeded in separating and collecting a novel DNase A which has the substrate specificity of recognizing the base sequence in double-stranded DNA molecules and cleaving the strands at specific sites in the DNA to produce specific DNA fragments, from the cell-free extract obtained by culturing Saccharomycetaceae such as Saccharomyces uvarum or Saccharomyces cerevisiae which belongs to the genus Saccharomyces, and Pichia membranaefaciens which belongs to the genus Pichia. The inventors established the process for production of the DNase A and completed the present invention.